The experiments outlined in this proposal are designed to delineate the early period of Sindbis virus replication. Use of conditions which inhibit RNA synthesis will allow us to define viral activities as "early" or "late" based on their expression in the absence of RNA synthesis. Specifically, we intend to determine the intracellular location and stability of the parental RNA during early eclipse phase, and to determine the nature and kinetics of induction of proteins and activities specified by the parental RNA. Induction of homologous interference, inhibition of host cell macromolecular synthesis, synthesis of viral polymerases and inhibition of cell division will be investigated. These studies will be facilitated by our finding that adenosine nucleotides will reversibly inhibit viral RNA synthesis. We also have isolated a Sindbis mutant, SB-RL, which has altered growth characteristics in vitro and exhibits reduced virulence in suckling mice when compared to wild-type. We will be examining the molecular basis for the reduced pathogenicity of SB-RL.